Calcium (Ca2+) signaling requires Ca2+ concentration varying with time. Calmodulin (CaM) is a main target for decoding the Ca2+ signal but its intrinsic Ca2+-binding properties alone appear insufficient to decode rapidly fluctuating Ca2+ signals. We propose that in addition to transducing the signal downstream, CaM-targets directly tune the Ca2+-binding properties of CaM through reciprocal interactions mediated by conformational adjustments that add an undiscovered temporally varying mechanism for producing target selectivity. Further, we propose that the target induced tuning of CaM's Ca2+-binding properties from the perspective of ter- molecular reactions is the missing piece to the puzzle for how CaM selectivity is mediated. The objective of the present proposal is to develop a novel approach characterizing how CaM binds Ca2+ and its target protein reciprocally, which underlies the central feature of CaM's target selectivity. Work accomplished in the previous funding period has shown that CaM binding to its targets is a process involving conformationally and mutually induced fit and that the conformational adjustments in both CaM and target molecules must be overcome in the pathway of binding. Our progress has built the foundation for the central hypothesis in this continuing project that the Ca2+ in a Ca2+-binding loop tunes the local electric field and the loop conformations differentially among the four EF-hands of CaM. The integrative dynamics of these EF hands adjusts the reciprocal relations between CaM's Ca2+ binding and target binding, which is distinctive to a target. We further demonstrate the principle of target selectivity with a system of two antagonist targets, neurogranin and CaM-dependent kinase II, interacting with CaM as a module for decoding distinct patterns of Ca2+ input. The rationale is that once the principle of tuning the reciprocal relation by a protein target is identified, we can design such protein targets that control CaM to respond to different frequencies or amplitudes of Ca2+ for transducing signals in live cells. We will test our central hypothesis by pursuing the following three thrusts: (1) How does the four EF hands of CaM differentially react to a target protein and shape the global conformation of CaM under the variable content of Ca2+-binding? (2) How does Ca2+ tune its local electric field and conformations of an EF hand in response to target binding? (3) How does CaM select from the two competing protein targets that tune CaM's affinity for Ca2+ in opposite directions? The research proposed is innovative because we will develop a novel computer model for Ca2+ sensing in CaM by integrating quantum mechanical calculations, molecular simulations, and biophysical and biochemical experiments, to characterize how target selectivity can be achieved. The proposed research is of significance because the proposed study would engender a breakthrough in our understanding of these processes and will provide insights into how the time and amplitude varying Ca2+ signal is decoded by CaM and the family of CaM-binding targets into coordinated biological responses.